Tetrahydropyrrolopyrimidinediones And Their Use In Therapy

ABSTRACT

The subject invention provides compounds of formula (I): 
     
       
         
         
             
             
         
       
     
     Including monomers and multimers thereof that are inhibitors of human neutrophil elastase (HNE) activity and are useful in the treatment of diseases or conditions in which HNE plays a part.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/264,621, filed Nov. 4, 2008; which is a continuation-in-partapplication of International Application No. PCT/GB2007/001638, filedMay 3, 2007, which claims priority to Great Britain Applications Nos.0608844.7, filed May 4, 2006 and 0612544.7, filed Jun. 23, 2006. Thisapplication is also a continuation-in-part of International ApplicationNo. PCT/GB2007/002825, filed Jul. 25, 2007 and PCT/GB2008/055439, filedMay 2, 2008, all of which are incorporated herein in their entirety.

FIELD OF THE INVENTION

This invention relates to heterocyclic compounds which are substituted3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine-2,5-diones, and their usein therapy.

BACKGROUND TO THE INVENTION

Human neutrophil elastase (NNE) is a 32 kDa serine proteinase found inthe azurophilic granules of neutrophils. It has a role in thedegradation of a wide range of extracellular matrix proteins, includingfibronectin, laminin, proteoglycans, Type III and

Type IV collagens as well as elastin (Bieth, G. In Regulation of Matrixaccumulation, Mechem. R. P. (Eds), Academic Press, NY, USA 1986,217-306). HNE has long been considered to play an important role inhomeostasis through repair and disposal of damaged tissues viadegradation of the tissue structural proteins. It is also relevant inthe defence against bacterial invasion by means of degradation of thebacterial body. In addition to its effects on matrix tissues, HNE hasbeen implicated in the upregulation of IL-8 gene expression and alsoinduces IL-8 release from the epithelial cells of the lung. In animalmodels of Chronic Obstructive Pulmonary Disease induced by tobacco smokeexposure both small molecule inhibitors and protein inhibitors of HNEinhibit the inflammatory response and the development of emphysema(Wright, J. L. et al. Am. J. Reap/r. Crit. Care Med. 2002, 166, 954-960;Churg, A. et al. Am. J. Respir. Crit, Care Med. 2003, 168, 199-207).Thus, HNE may play a role both in matrix destruction and in amplifyinginflammatory responses in chronic respiratory diseases where neutrophilinflux is a characteristic feature. Indeed, HNE is believed to play arole in several pulmonary diseases, including chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), acute respiratorydistress syndrome (ARDS), pulmonary emphysema, pneumonia and lungfibrosis. It is also implicated in several cardiovascular diseases inwhich tissue remodelling is involved, for example, in heart failure andthe generation of ischaemic tissue injury following acute myocardialinfarction.

COPD is an umbrella term encompassing three different pathologicalconditions, all of which contribute to limitation of airflow: chronicbronchitis, emphysema and small-airway disease. Generally all three willexist to varying extents in patients presenting with COPD, and all threemay be due to neutrophil-mediated inflammation, as supported by theincreased number of neutrophils observed in bronchoalveolar leakage(BAL) fluids of COPD patients (Thompson, A. B.; Daughton, D.; et al. Am.Rev. Respir. Dis. 1989, 140, 1527-1537). The major pathogenicdeterminant in COPD has long been considered to be theprotease-anti-protease balance (also known as the‘elastase:anti-elastase hypothesis’), in which an imbalance of HNE andendogenous antiproteases such as a 1-antitrypsin (a₁-AT),

Secretory leukocyte protease inhibitor (SLP!) and pre-elafin leads tothe various inflammatory disorders of COPD. Individuals that have agenetic deficiency of the protease inhibitor al-antitrypsin developemphysema that increases in severity over time (Laurrell, C. B.;Erikkson, S Scand. J. Clin. Invest. 1963 15, 132-140). An excess of HNEis therefore destructive, leading to the breakdown of pulmonarymorphology with loss of elasticity and destruction of alveolarattachments of airways in the lung (emphysema) whilst simultaneouslyincreasing microvascular permeability and mucus hypersecretion (chronicbronchitis).

Multimeric ligands consist of multiple binding domains which aretethered together through a suitable scaffold. Hence individual bindingdomains are linked together into a single molecule, increasing theprobability that the multinner will bind sequentially in a step-wisemanner with multiple active sites resulting in high-affinityinteractions (Handl, H. L. et al. Expert Opin. Ther. Targets 2004, 8,565-586; Han, Y. F. et al., Bioorg. Med. Chem. Letts. 1999, 7,2569-2575). Also, multiple binding interactions (either sequential orparallel) with relatively high off-rates can combine to yield an overalllow off-rate for the multimeric ligand. Thus, a molecule consisting of asuitable linker and ligands may be expected to show advantage over themonomeric ligands alone in terms of potency and/or duration of action.Multimeric compounds are unlikely to be orally bioavailable (aspredicted by Lipinski's “Rule of 5”) which may be advantageous where aninhaled route of administration to the lungs is targeted, since evenafter inhaled administration, a large proportion of drug is likely toenter the GI tract. Thus such compounds may be expected to show reducedsystemic exposure after inhalation administration and hence an improvedtoxicity profile over orally administered therapies.

BRIEF DESCRIPTION OF THE INVENTION

This invention provides novel compounds which are inhibitors of HNE, andare useful in the treatment of diseases or conditions in which HNEactivity plays a part. The compounds of the invention may be used asmonomers or, particularly in the case of topical pulmonary applicationby inhalation, in the form of multimers, such as dimers, covalentlylinked via a linker framework.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides a compound of formula (I),:

wherein

A is aryl or heteroaryl;

D is oxygen or sulphur;

R¹, R², R³ and R⁵ are independently each hydrogen, halogen, nitro,cyano, C₁-C₆-alkyl, C₂-C₆-alkenyl, C₂-C₆-alkynyl, hydroxy orC₁-C₆-alkoxy or C₂-C₆-alkenyloxy, wherein C₁-C₆-alkyl andC_(i)-C₆-alkoxy can be further substituted with one to three identicalor different radicals selected from the group consisting of halogen,hydroxy and C₁-C₄-alkoxy;

R and R⁴ each independently represent a radical of formula-[X]_(m)-[Alk¹]_(p)-[Q]_(n)-[Alk²]_(Q)-[X¹]_(k)-Z wherein

k, m, n, p and q are independently 0 or 1;

Alk¹ and Alk² each independently represent an optionally substitutedC₁-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally containan ether (—O—), thioether (—S—) or amino (—NR^(A)—) link wherein R^(A)is hydrogen or C₁-C₃ alkyl;

Q represents (i) —O—, —S—, —S(═O)—, —S(═O)₂—, —S⁺(R^(A))—, —N(R^(A))—,—N⁺(R^(A))(R^(B))—, —C(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)NR^(A)—,—NR^(A)C(═O)—, —S(O₂)NR^(A)—, —NR^(A)S(O₂)—, —NR^(A)C(═O)NR^(B),—NR^(A)C(═NR^(A))NR^(B), —C(═NR^(D))NR^(E), —NR^(E)C(═NR^(D))—,whereinR^(A), R^(B), R^(D) and R^(E) are independently hydrogen, C₁-C₆ alkyl,or C₃-C₆ cycloalkyl, or R^(A) and R^(B), or R^(D) and R^(E) takentogether with the nitrogen to which they are attached form a monocyclicheterocyclic ring of 5 to 7 ring atoms which my contain a furtherheteroatom selected from N, O and S, or (ii) an optionally substituteddivalent mono- or bicyclic carbocyclic or heterocyclic radical having3-6 ring members;

X represents —(C═O)—, —S(O₂)—, —C(═O)O—, —(C═O)NR^(A)—, or—S(O₂)NR^(A)—, wherein R^(A) is hydrogen. C₁-C₆ alkyl, or C₃-C₆cycloalkyl;

X¹ represents —O—, —S—, or —NH; and

Z is hydrogen or an optionally substituted mono- or bicyclic carbocyclicor heterocyclic radical having 3-6 ring members.

The invention also includes a multimeric compound comprising two, threeor four molecules of a compound of formula (I) above, covalently linkedthrough a linker framework.

Compounds of formula (I) above and multimers thereof may be prepared inthe form of salts, particularly pharmaceutically acceptable salts,N-oxides, hydrates and solvates thereof. Any claim to a compound herein,or reference to “compounds of the invention”, compounds with which theinvention is concerned”, compounds of formula (I), and the like includessalts. N-oxides, hydrates and solvates of such compounds.

Compounds of the invention may be useful in the treatment or preventionof diseases in which HNE is implicated, for example chronic obstructivepulmonary disease (COPD), chronic bronchitis, lung fibrosis, pneumonia,acute respiratory distress syndrome (ARDS), pulmonary emphysema,smoking-induced emphysema and cystic fibrosis.

Hence other aspects of the invention are (i) a pharmaceuticalcomposition comprising a compound of the invention and apharmaceutically acceptable carrier or excipient; and (ii) the use of acompound of the invention for the manufacture of a medicament for thetreatment or prevention of a disease or condition in which HNE isimplicated.

Terminology

As used herein, the term “(C_(a)-C_(b))alkyl” wherein a and b areintegers refers to a straight or branched chain alkyl radical havingfrom a to b carbon atoms. Thus when a is 1 and b is 6, for example, theterm includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term “(C_(a)-C_(b))alkenyl” wherein a and b areintegers refers to a straight or branched chain alkenyl moiety havingfrom a to b carbon atoms having at least one double bond of either E orZ stereochemistry where applicable. Thus when a is 2 and b is 6, forexample, the term includes, for example, vinyl, allyl, 1- and 2-butenyland 2-methyl-2-propenyl.

As used herein the term “C_(a)-C_(b) alkynyl” wherein a and b areintegers refers to straight chain or branched chain hydrocarbon groupshaving from a to b carbon atoms and having in addition one triple bond.Thus when a is 1 and b is 6, for example, the term includes for example,ethynyl (—C═CH), 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and5-hexynyl.

As used herein the term “divalent (C_(a)-C_(b))alkylene radical” whereina and b are integers refers to a saturated hydrocarbon chain having froma to b carbon atoms and two unsatisfied valences.

As used herein the term “divalent (C_(a)-C_(b))alkenylene radical”wherein a and b are integers refers to a divalent hydrocarbon chainhaving from 2 to 6 carbon atoms, and at least one double bond.

As used herein the unqualified term “carbocyclic” refers to a mono-, bi-or tricyclic radical having up to 16 ring atoms, all of which arecarbon, and includes aryl and cycloalkyl.

As used herein the unqualified term “cycioalkyl” refers to a monocyclicsaturated carbocyclic radical having from 3-8 carbon atoms and includes,for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

As used herein the unqualified term “aryl” refers to a mono-, bi- ortri-cyclic carbocyclic aromatic radical, and includes radicals havingtwo monocyclic carbocyclic aromatic rings which are directly linked by acovalent bond. Illustrative of such radicals are phenyl, biphenyl andnapthyl.

As used herein the unqualified term “heteroaryl” refers to a mono-, bi-or tri-cyclic aromatic radical containing one or more heteroatomsselected from S, N and O, and includes radicals having two suchmonocyclic rings, or one such monocyclic ring and one monocyclic arylring, which are directly linked by a covalent bond. Illustrative of suchradicals are thienyl, benzthienyl, furyl, benzfuryl, pyrrolyl,imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl,benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl,benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl,oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,indolyl and indazolyl.

As used herein the unqualified term “heterocyclyl” or “heterocyclic” or“heterocycloalkyl” includes “heteroaryl” as defined above, and in itsnon-aromatic meaning relates to a mono-, bi- or tri-cyclic non-aromaticradical containing one or more heteroatoms selected from S, N and O, andto groups consisting of a monocyclic non-aromatic radical containing oneor more such heteroatoms which is covalently linked to another suchradical or to a monocyclic carbocyciic radical. Illustrative of suchradicals are pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl,pyrrolidinyl, pyrimidinyl, morphoiinyl, piperazinyl, indolyl,morpholinyl, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl,methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimidogroups.

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with upto four compatible substituents, each of which independently may be, forexample, (C₁-C₆)alkyl, cycloalkyl, (C₁-C₆)alkoxy, hydroxy,hydroxy(C₁-C₆,)alkyl, mercapto, mercapto(C₁-C₆)alkyl, (C₁-C₆)alkylthio,phenyl, monocyclic heteroaryl having 5 or 6 ring atoms, halo (includingfluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy, nitro,nitrite (—CN), oxo, —COOH, —COOR^(A), —COR^(A), —SO₂R^(A), —CONH₂,—SO₂NH₂, —CONHR^(A), —SO₂NHR^(A), —CONR^(A)R^(B), —SO₂NR^(A)R^(B), —NH₂,—NHR^(A), —NR^(A)R^(B), —OCONH₂, —OCONHR^(A), —OCONR^(A)R^(B),—NHCOR^(A), —NHCOOR^(A), —NR^(B)COOR^(A), —NHSO₂OR^(A), —NR^(B)SO₂OH,—NR^(B)SO₂OR^(A), —NHCONH₂, —NR^(A)CONH₂, —NHCONHR^(B) —NR^(A)CONHR^(B),—NHCONR^(A)R^(B), or —NR^(A)CONR^(A)R^(B) wherein R^(A) and R^(B) areindependently a (C₁-C₆)alkyl, (C₃-C₆) cycloalkyl , phenyl or monocyclicheteroaryl having 5 or 6 ring atoms, or R^(A) and R^(B) when attached tothe same nitrogen atom form a cyclic amino ring, such as piperidinyl,morpholinyl or piperazinyl. An “optional substituent” may be one of theforegoing substituent groups.

As used herein the term “salt” includes base addition, acid addition andquaternary salts. Compounds of the invention which are acidic can formsalts, including pharmaceutically acceptable salts, with bases such asalkali metal hydroxides, e.g. sodium and potassium hydroxides; alkalineearth metal hydroxides e.g. calcium, barium and magnesium hydroxides;with organic bases e.g. N-methyl-D-glucamine, cholinetris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethylpiperidine, dibenzylamine and the like. Those compounds (I) which arebasic can form salts, including pharmaceutically acceptable salts withinorganic acids, e.g. with hydrohalic acids such as hydrochloric orhydrobromic acids, sulphuric acid, nitric acid or phosphoric acid andthe like, and with organic acids e.g. with acetic, tartaric, succinic,fumaric, maleic, malic, salicylic, citric, methanesulphonic,p-toluenesulphonic, benzoic, benzenesunfonic, glutamic, lactic, andmandelic acids and the like. Those compounds (I) which have a basicnitrogen can also form quaternary ammonium salts with a pharmaceuticallyacceptable counter-ion such as chloride, bromide, acaetate, formate,p-toluenesulfonate, succinate, hemi-succinate, naphthalene-bissulfonate, methanesulfonate, xinafoate, and the like.

Compounds of the invention which contain one or more actual or potentialchiral centres, because of the presence of asymmetric carbon atoms, canexist as a number of diastereoisomers with R or S stereochemistry ateach chiral centre. The invention includes all such diastereoisomers andmixtures thereof.

In the monomeric compounds of the invention of formula (I), in anycompatible corn bin ation:

The atom D may be O or S. O is currently preferred.

The ring A is aryl or heteroaryl and may be any of those rings listedabove as examples of aryl or heteroaryl, especially phenyl andmonocyclic heteroaryl having 5 or 6 ring atoms. Specific exampiesinclude pyridyl, such as 2- and 3-pyridyl, or pyrimidinyl such aspyrirnidin-2-yl, but presently it is preferred that A be phenyl.

R¹ and R² may be selected from any of the substituent types for whichthey are defined in relation to formula (I), including hydrogen,halogen, nitro, cyano, C₁-C₃-alkyl, C₂-C₃-alkenyl, C₂-C₃-alkynyl,hydroxy or C₁-C₃-alkoxy or C₂-C₃-alkenyloxy. Specific examples of suchsubstitutuents include hydrogen, fluoro, chloro, bromo, cyano, methyl,methoxy and —C═CH. For example, —AR¹R² may be 4-cyanophenyl or4-ethynylphenyl.

R and R⁵ too may be selected from any of the substituent types for whichthey ³ and R⁵ are defined in relation to formula (I), but in onecurrently preferred type of compound of the invention R³ and R⁵ areindependently H, CF₃, F, Cl or Br. The preferred position ofsubstitution on the phenyl ring by R³ and/or R⁵ is 3-, 4- or 5-.

Presently it is believed that the monomers of the invention can interactwith HNE as inhibitors with the R or R⁴ substituent located remote fromthe binding interface, extending towards solvent. Hence those groupsprovide sites for modulation of solubility and other pharmacokineticproperties. Accordingly R and R⁴ may vary widely, and are defined inrelation to formula (I) as a radical of formula-[X],-[Alk¹]_(p)-[Q],-[Alk²]_(q)-[X¹]_(k)-Z. According to thatdefinition, k, m, n, p and q may all be 0, and Z may be hydrogen, sothat R or R⁴ itself may be hydrogen. However, many other classes of R orR⁴ substituent are encompassed by selecting different combinations ofvalues for the variables.

For example R or R⁴ may be selected from C₁-C₆-alkyl, formyl,aminocarbonyl, mono- or di-C₁-C₄-alkylaminocarbonyl,C₃-C₈-cycloalkylcarbonyl, C₁-C₆-alkylcarbonyl, C₁-C₆-alkoxycarbonyl,N-(C₁-C₄-alkylsulfonyl)-aminocarbonyl,N-(C₁-C₄-alkylsulfonyl)-N-(C₁-C₄-alkyl)-aminocarbonyl, heteroaryl,heterocycloalkyl, heteroarylcarbonyl or heterocycloalkylcarbonyl;wherein C₁-C₆-alkyl, mono- and di-C₁-C₄-alkylaminocarbonyl,C₁-C₆-alkylcarbonyl. C₁-C₆-alkoxycarbonyl, heteroaryl andheterocycloalkyl can be substituted with one to three identical ordifferent radicals selected from the group consisting of aryl,heteroaryl, hydroxyl, C₁-C₄-alkoxy, hydroxycarbonyl,C₁-C₆-alkoxycarbonyl, aminocarbonyl, mono anddi-C₁-C₄-alkylaminocarbonyl, amino, mono- and di-C₁-C₄-alkylamino.C₁-C₄-alkylcarbonylamino, cyano. N-(mono- anddi-C₁-C₄-alkylamino-C₁-C₄-alkyl)-aminocarbonyl.N-(C₁-C₄-alkoxy-C₁-C₄-alkyl)-aminocarbonyl and halogen.

In a particular subclass of compounds of the invention, R⁴ and/or R isradical of formula -[X]_(m)-[Alk¹]_(p)-[Q]_(n)-[Alk²]_(q)-[X¹]_(k)-Zwherein m is 0, and k, p, n and q are each 1, Q is —N(R^(A)) or—N⁺(R^(A))(R^(B))—, and R^(A), R^(B) Alk¹, Alk², X₁ and Z are as definedin relation to formula (I). In this subclass. X¹ may be, for example,—O—, and Z may be, for example optionally substituted phenyl ormonocyclic hetroaryl, the latter having 5 or 6 ring atoms.

In the compounds of the invention one of R and R⁴ may be hydrogen, whilethe other is a substitutent other than hydrogen

Other types of R and R⁴ groups have Formula (VIIIA), (VIIIB) or (VIIIC):

wherein

R^(4A) is hydrogen or C₁-C₆-alkyl, and s is 1 or 2.

Further types of R and R⁴ groups have Formula (IX)

wherein

R^(4B) is hydrogen or C₁-C₆-alkyl;

R^(4C), R^(4D), R^(4E) are each C₁-C₆-alkyl, and the nitrogen to whichthey are attached is quaternary and carries a positive charge; andadditionally any two of R^(4B), R^(4D), R^(4E) may be joined to form aring, optionally containing a second heteroatom selected from oxygen ornitrogen;

or

One of R^(4C), R^(4D), R^(4E) is a lone pair and the other groups are asdefined above, and the nitrogen to which they are attached is tertiary;and

v1 and v2 are each independently 0-5.

Other types of R and R⁴ groups are those selected from the following:

wherein

R^(4B) is hydrogen or C₁-C₆-alkyl;

R^(4C), R^(4D),R^(4E) are each C₁-C₆-alkyl, and the nitrogen to whichthey are attached is quaternary and carries a positive charge; andadditionally any two of R^(4C), R^(4D), R^(4E) may be joined to form aring, optionally containing a second heteroatom selected from oxygen ornitrogen;

or

one of R^(4C), R^(4D, R) ^(4E) is a lone pair and the other groups areas defined above, and the nitrogen to which they are attached istertiary;

R^(4F) and R^(4I) are independently hydrogen or C₁-C₆-alkyl;

R^(4G) and R^(4H) are independently hydrogen or C₁-C₆-alkyl, or R^(4G)and R^(4H) taken together with the nitrogen to which they are attachedform a monocyclic heterocyclic ring of 5 to 7 ring atoms which mycontain a further heteroatom selected from N, O and S; and

v1 and v2 are each independently 0-5.

In the multimeric compounds of the invention, two, three or fourmolecules of a monomeric compound of the invention are covalently linkedthrough a linker framework. Since the linker framework need not play anactive role in interacting with the HNE enzyme, its role is simply toallow binding contact between one or more of the monomeric elements andthe enzyme. Hence a vast range of chemistries may be envisaged for thelinker framework. Furthermore, the point of attachment of the monomericelements to the linker framework may be selected according to theparticular linker chemistry to be employed. Presentiy it is preferredthat two, three or four of the monomeric molecules are linked to thelinker framework via their respective nitrogen atoms shown in formula(I) as linked to R or R⁴.

Furthermore, it is presently preferred that only two of the monomers areso linked. In the latter case, the linker framework may be, for example,the linker framework may be a divalent straight chain, saturated orunsaturated hydrocarbon radical having from 2 to 12 carbon atoms in thesaid chain, and wherein one or more carbons may be replaced by adivalent monocyclic or bicyclic carbocyclic or heterocyclic radicalhaving from 3 to 7 ring atoms in the or each ring, or by —O—, —S—,—S(═O)—, —S(═O)₂—, —C(═O)—, —N(R^(P))—, —N⁺(R^(P))(R^(Q))—, —C(═O)O—,—OC(═O)—, —C(═O)NR^(A)—, —NR^(A)C(═O)—, —S(O₂)NR^(A)—, —NR^(A)S(O₂)—,—NR^(A)C(═O)NR^(B)—, —NR^(A)C(═NR^(A))NR^(B)—, —C(═NR^(D))NR^(E)—, or—NR^(E)C(═NR^(D))—, wherein R^(A), R^(B), R^(D) and R^(E) areindependently hydrogen. C₁-C₆ alkyl, or C₃-C₆ cycloalkyl, and R^(P) andR^(Q) are independently hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl,HO—(C₁-C₆ alkyl)-, R^(A)R^(B)N—(C₁-C₆ alkyl)-, or HOC(═O)—(C₁-C₆alkyl)-, or R^(A) and R^(B), or R^(D) and R^(E), or R^(P) and R^(Q)taken together with the nitrogens to which they are attached form amonocyclic heterocyclic ring of 5 to 7 ring atoms which may contain afurther heteroatom selected from N, O and S.

When one or more one or more —(CH₂)— groups of the linker framework isor are replaced by a divalent monocyclic or bicyclic carbocyclic orheterocyclic radical, the said radical may be selected from, forexample, the following:

The linker framework may have, for example, one of the followingstructures (A), (B),(C), (D), (E), (G) and (E):

Specific linker frameworks of the above type include those present inthe dimer compounds of the examples herein.

Thus, one preferred subset of the multimers of the invention has theformula M-L-M¹ wherein L is a divalent linker radical, for example ofthe kinds discussed above as linker frameworks, and M and M¹ are eachindependently a radical of formula (IA) wherein D, A and R¹-R⁵ are asdefined and discussed above:

Preferably also, M and M¹ are the same.

Another preferred subset of the multimers of the invention has theformula M-L-M¹ wherein L is a divalent linker radical for example of thekinds discussed above as linker frameworks, and M and M¹ are eachindependently a radical of formula (IB) wherein D, A and R, R¹, R², R³and R⁵ are as defined and discussed above:

Here too, it is currently preferred that M and M¹ are the same.

Specific examples of such dimeric compounds of formula (IA) and (IB)include those of the Examples herein.

The therapeutic utility of the present compounds is pertinent to anydisease that is known to be at least partially mediated by the action ofhuman neutrophil elastase. For example, the present compounds may bebeneficial in the treatment of chronic obstructive pulmonary disease(COPD), cystic fibrosis (CF), acute respiratory distress syndrome(ARDS), pulmonary emphysema, pneumonia and lung fibrosis.

The present invention is also concerned with pharmaceutical formulationscomprising, as an active ingredient, a compound of the invention. Othercompounds may be combined with compounds of this invention for theprevention and treatment of inflammatory diseases of the lung. Thus thepresent invention is also concerned with pharmaceutical compositions forpreventing and treating inflammatory diseases of the lung comprising atherapeutically effective amount of a compound of the invention and oneor more other therapeutic agents.

Suitable therapeutic agents for a combination therapy with compounds ofthe invention include: (1) a corticosteroid, for example fluticasone orbudesonide; (2) a) β2-adrenoreceptor agonist, for example salmeterol orformeterol; (3) a leukotriene modulator, for example montelukast orpraniukast; (4) anticholinergic agents, for example selectivemuscarinic-3 (M3) receptor antagonists such as tiotropium bromide; (5)phosphodiesterase-IV (PDE-IV) inhibitors, for example roflumilast orcilomilast; (6) an antitussive agent, such as codeine or dextramorphan;and (7) a non-steroidal anti-inflammatory agent (NSAID), for exampleibuprofen or ketoprofen.

The weight ratio of the first and second active ingredients may bevaried and will depend upon the effective dose of each ingredient.Generally, an effective dose of each will be used.

The magnitude of prophylactic or therapeutic dose of a compound of theinvention will, of course, vary with the nature of the severity of thecondition to be treated and with the particular compound and its routeof administration, and will generally be determined by clinical trial asrequired in the pharmaceutical art. It will also vary according to theage, weight and response of the individual patient. In general, thedaily dose range will lie within the range of from about 0.001 mg toabout 100 mg per kg body weight of a mammal, preferably 0.01 mg to about50 mg per kg, and most preferably 0.1 to 10 mg per kg, in single ordivided doses. On the other hand, it may be necessary to use dosagesoutside these limits in some cases.

Another aspect of the present invention provides pharmaceuticalcompositions which comprise a compound of the invention and apharmaceutically acceptable carrier. The term “composition”, as inpharmaceutical composition, is intended to encompass a productcomprising the active ingredient(s), and the inert ingredient(s)(pharmaceutically acceptable excipients) that make up the carrier, aswell as any product which results, directly or indirectly, fromcombination, complexation or aggregation of any two or more of theingredients, or from dissociation of one or more of the ingredients, orfrom other types of reactions or interactions of one or more of theingredients. Accordingly, the pharmaceutical compositions of the presentinvention encompass any composition made by admixing a compound of theinvention, additional active ingredient(s), and pharmaceuticallyacceptable excipients.

The pharmaceutical compositions of the present invention comprise acompound of the invention as an active ingredient or a pharmaceuticallyacceptable salt thereof, and may also contain a pharmaceuticallyacceptable carrier and optionally other therapeutic ingredients. Theterm “pharmaceutically acceptable salts” refers to salts prepared frompharmaceutically acceptable non-toxic bases or acids including inorganicbases or acids and organic bases or acids.

Any suitable route of administration may be employed for providing amammal, especially a human, with an effective dosage of a compound ofthe present invention. in therapeutic use, the active compound may beadministered by any convenient, suitable or effective route. Suitableroutes of administration are known to those skilled in the art, andinclude oral, intravenous, rectal, parenteral, topical, ocular, nasal,buccal and pulmonary. Delivery by inhalation is preferred.

Compositions suitable for administration by inhalation are known, andmay include carriers and/or diluents that are known for use in suchcompositions, The composition may contain 0.01-99% by weight of activecompound. Preferably, a unit dose comprises the active compound in anamount of 1 μg to 10 mg.

The most suitable dosage level may be determined by any suitable methodknown to one skilled in the art. It will be understood, however, thatthe specific amount for any particular patient will depend upon avariety of factors, including the activity of the specific compound thatis used, the aae, body weight, diet, general health and sex of thepatient, time of administration, the route of administration, the rateof excretion, the use of any other drugs, and the severity of thedisease undergoing treatment.

For delivery by inhalation, the active compound is preferably in theform of microparticies. They may be prepared by a variety of techniques,including spray-drying, freeze-drying and micronisation.

By way of example, a composition of the invention may be prepared as asuspension for delivery from a nebuliser or as an aerosol in a liquidpropellant, for example for use in a pressurised metered dose inhaler(PMDI). Propellants suitable for use in a PMDI are known to the skilledperson, and include CFC-12, HFA-134a, HFA-227, HCFC-22 (CCl2F2) andHFA-152 (CH4F2 and isobutane).

In a preferred embodiment of the invention, a composition of theinvention is in dry powder form, for delivery using a dry powder inhaler(DPI). Many types of DPI are known.

Microparticles for delivery by administration may be formulated withexcipients that aid delivery and release. For example, in a dry powderformulation, microparticles may be formulated with large carrierparticles that aid flow from the DPI into the lung. Suitable carrierparticles are known, and include lactose particles; they may have a massmedian aerodynamic diameter of greater than 90 μm.

In the case of an aerosol-based formulation, a preferred composition is:

Compound of the invention   24 mg/canister Lecithin, NF Liq. Conc.  1.2mg/canister Trichlorofluoromethane, NF 4.025 g/canisterDichlorodifluoromethane, NF 12.15 g/canister.

Compounds of the invention may be used in combination with other drugsthat are used in the treatment/prevention/suppression or amelioration ofthe diseases or conditions for which present compounds are useful. Suchother drugs may be administered, by a route and in an amount commonlyused therefore, contemporaneously or sequentially with a compound of theinvention. When a compound of the invention is used contemporaneouslywith one or more other drugs, a pharmaceutical composition containingsuch other drugs in addition to the compound of the invention ispreferred. Accordingly, the pharmaceutical compositions of the presentinvention include those that also contain one or more other activeingredients, in addition to a compound of the invention.

The agents of the invention may be administered in inhaled form. Aerosolgeneration can be carried out using, for example, pressure-driven jetatomizers or ultrasonic atomizers, preferably using propellant-drivenmetered aerosols or propellant-free administration of micronized activecompounds from, for example, inhalation capsules or other “dry powder”delivery systems.

The active compounds may be dosed as described depending on the inhalersystem used. in addition to the active compounds, the administrationforms may additionally contain excipients, such as, for example,propellants (e.g. Frigen in the case of metered aerosols),surface-active substances, emulsifiers, stabilizers, preservatives,flavorings, fillers (e.g. lactose in the case of powder inhalers) or, ifappropriate, further active compounds.

For the purposes of inhalation, a large number of systems are availablewith which aerosols of optimum particle size can be generated andadministered, using an inhalation technique which is appropriate for thepatient. In addition to the use of adaptors (spacers, expanders) andpear-shaped containers (e.g. Nebulator®, Volumatic®), and automaticdevices emitting a puffer spray (Autohaler®), for metered) aerosols, inparticular in the case of powder inhalers, a number of technicalsolutions are available (e.g. Diskhaier®, Rotadisk®, Turbohaler® or theinhalers for example as described EP-A-0505321).

Methods of Synthesis

The compounds of the present invention can be prepared according to theprocedures of the following schemes and examples, using appropriatematerials, and are further exemplified by the following specificexamples. Moreover, by utilising the procedures described with thedisclosure contained herein, one of ordinary skill in the art canreadily prepare additional compounds of the present invention claimedherein. The compounds illustrated in the examples are not, however, tobe construed as forming the only genus that is considered as theinvention. The examples further illustrate details for the preparationof the compounds of the present invention. Those skilled in the art willreadily understand that known variations of the conditions and processesof the following preparative procedures can be used to prepare thesecompounds.

The compounds of the invention may be isolated in the form of theirpharmaceutically acceptable salts, such as those described previouslyherein above. The free acid or base form corresponding to isolated saltscan be generated by neutralisation with a suitable base or acid such assodium hydroxide, potassium carbonate, acetic acid and hydrochloric acidand extraction of the liberated free acid or base into an organicsolvent followed by evaporation. The free form isolated in this mannercan be further converted into another pharmaceutically acceptable saltby dissolution in an organic solvent followed by addition of theappropriate acid or base and subsequent evaporation, precipitation, orcrystallisation.

It may be necessary to protect reactive functional groups (e.g. hydroxy,amino, thio or carboxy) in intermediates used in the preparation ofcompounds of the invention to avoid their unwanted participation in areaction leading to the formation of the compounds. Conventionalprotecting groups, for example those described by T. W. Greene and P. G.M. Wuts in “Protective groups in organic chemistry” John Wiley and Sons,1999, may be used.

Compounds of the invention may be prepared according to the routesillustrated in Schemes 1 and 2.

The following Examples illustrate the invention.

General Experimental Details:

All solvents and commercial reagents were used as received. Whereproducts were purified using an solute™ SPE Si II cartridge, ‘IsoluteSPE Si cartridge’ refers to a pre-packed polypropylene column containingunbonded activated silica with irregular particles with average size of50 μm and nominal 60 Å porosity. Where an Isolute™ SCX-2 cartridge wasused, ‘Isolute SCX-2 cartridge’ refers to a pre-packed polypropylenecolumn containing a non end-capped propyisulphonic acid functionalisedsilica strong cation exchange sorbent. ‘Isolute Al—N cartridge’ refersto a pre-packed polypropylene column containing neutral alumina withaverage particle size 50-200 μm and 120 Å pore diameter.

Preparative HPLC Conditions: HPLC System 1:

C18-reverse-phase column (100×22.5 mm i.d Genesis column with 7 μmparticle size), eluting with a gradient of A: water+0,1% formic acid; B:acetonitrile+0.1% formic acid at a flow rate of 5 ml/min and gradient of1%/min increasing in B. UV detection at 230 nm. Compounds were obtainedas the formate salt where stated.

HPLC System 2:

C18-reverse-phase end-capped column (250×21.2 mm Gemini column with 5 μmparticle size), eluting with a gradient of A: water+0.1% formic acid; B:acetonitrile+0.1% formic acid with a flow rate typically 17 ml/min andgradient of 1%/min increasing in B. UV detection at 254 nm, Compoundswere obtained as the formate salt where stated.

HPLC System 3:

C18-reverse-phase end-capped column (250×21.2 mm Gemini column with 5 μmparticle size), eluting with a gradient of A: water; B: acetonitrilewith a flow rate typically 17 ml/min and gradient of 196/min increasingin B. UV detection at 254 nm.

HPLC System 4:

C18-reverse-phase end-capped column (250×21.2 mm Gemini column with 5 μmparticle size), eluting with a gradient of A: water; B: MeOH with a flowrate typically 17 ml/min and gradient of 1%/min increasing in B. UVdetection at 254 nm.

HPLC System 5:

C18-reverse-phase column (250×21.2 mm Luna column with 5 μm particiesize), eluting with a gradient of A: water+0.1% formic acid; B:acetonitrile+0.1% formic at a flow rate of 15 ml/rnin and gradient of1%/min increasing in B. UV detection at 254 nm. Compounds were obtainedas the formate salt where stated.

LC-MS Method 1:

Waters Platform LC with a C18-reverse-phase column (30×4.6 mm PhenomenexLuna 3 μm particle size), elution with A: water 0.1% formic acid; B:acetonitrile+0.1% formic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 2.0 95 5 0.50 2.0 95 5 4.50 2.05 95 5.50 2.0 5 95 6.00 2.0 95 5

Detection—MS, ELS, UV (100 μl split to MS with in-line UV detector)

MS ionisation method—Electrospray (positive and negative ion)

LC-MS Method 2:

Waters Micromass ZMD with a C18-reverse-phase column (30×4.6 mmPhenomenex Luna 3 μm particle size), elution with A: water+0.1% formicacid; B: acetonitrile+0.1% formic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 2.0 95 5 0.50 2.0 95 5 4.50 2.05 95 5.50 2.0 5 95 6.00 2.0 95 5

Detection—MS, ELS, UV (100 μl split to MS with in-line UV detector)

MS ionisation method—Electrospray (positive and negative ion)

LC-MS Method 3:

Micromass Platform LCT with a 018-reverse-phase column (100×3.0 mmHiggins Clipeus with 5 μm particle size), elution with A: water+0.1%formic acid; B: acetonitrile+0.1% formic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 1.0 95 5 1.00 1.0 95 5 15.001.0 5 95 20.00 1.0 5 95 22.00 1.0 95 5 25.00 1.0 95 5

Detection—MS, ELS, UV (100 μl split to MS with in-line UV detector)

MS ionisation method—Electrospray (positive ion)

LC-MS Method 4:

Waters Micromass ZQ2000 with a C18-reverse-phase column (100×3.0 mm

Higgins Clipeus with 5 μm particle size), elution with A: water+0.1%formic acid; B: acetonitrile+0.1% formic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 1.0 95 5 1.00 1.0 95 5 15.001.0 5 95 20.00 1.0 5 95 22.00 1.0 95 5 25.00 1.0 95 5

Detection—MS, ELS, UV (100 μl split to MS with in-line UV detector)

MS ionisation method—Electrospray (positive ion)

LC-MS Method 5:

Waters Platform LC quadrupole mass spectrometer linked to a HewlettPackard HP1100 LC system with a C18 reverse-phase column (30×4.6 mmPhenomenex Luna with 3 μm particle size), elution with A: water+0.1%formic acid; B: acetonitrile+0.1% formic acid. Gradient as for method 1.

LC-MS Method 6:

Waters Micromass ZQ2000 linked to a Hewlett Packard HP1100 LC systemwith a C18-reverse-phase column (100×3.0 mm Higgins Clipeus with 5 μmparticle size), elution with A: water+0.1% formic acid; B:acetonitrile+0.1% formic acid. Gradient as for method 3.

Abbreviations Used in the Experimental Section:

-   -   DOM=dichloromethane    -   DMF=N,N-dimethylformamide    -   HPLC=high performance liquid chromatography    -   IMS=industrial methylated spirits    -   RT=room temperature    -   Rt=retention time    -   THF=tetrahydrofuran    -   TFA=trifluoroacetic acid

The following intermediates can be prepared according to the referencegiven:

Inter- me- diate Structure Reference 1

WO2006/082412 2

WO2006/082412 3

WO2006/082412 4

WO2006/082412 5

WO2006/082412 6

WO2006/082412 7

WO2006/082412 8

WO2004/024700 9

WO2004/024700 10 

WO2004/024700 11 

WO2006/082412 12 

WO2006/082412 13 

WO2006/082412

Intermediate 14

Polyphosphoric and (17.2 g) was suspended in THF (90 ml) and stirredmechanically whilst 3.5-difluorophenylurea (5.40 g. 31.4 mmol),4-cyanobenzaldehyde (4.94 g, 37.6 mmol) and ethyl acetoacetate (3.97 ml,31.4 mmol) were added. The resulting mixture was heated at reflux for 17h, and then left at room temperature for 48 h. The solvent was removedunder reduced pressure and the residue partitioned between water andEtOAc. The organic layer was washed with water, aqueous sodium carbonatesolution, water then brine and dried (MgSO₄), filtered and concentrated.The resulting foam was purified in two batches on a Biotage™ flashchromatography cartridge (90 g), loading in DOM and eluting with17.5-20-25% EtOAc in iso-hexanes. The foam thus obtained was trituratedwith iso-hexanes/Et₂O, then collected as a white solid by filtration,subjected to one displacement wash with 2:1 iso-hexanes:Et₂O and driedin a vacuum oven.

Yield: 6.63 g (53%)

LC-MS (Method 1): Rt=3.55 min, m/z=398 [M+H]⁺

Intermediate 15

Intermediate 15 was prepared from 4-iodobenzaldehyde, ethyl acetoacetateand 3-(trifluoromethyl)phenylurea using a similar method to that used inthe preparation of Intermediate 14.

Yield: (25%)

LC-MS (Method 1): Rt=4.17 min, m/z=531 [M+H]⁺

Intermediate 16

Intermediate 1 (5.00 g, 11.7 mmol) was dissolved in chloroform (140 ml)and bromine (1.87 g, 11.7 mmol) was added dropwise with stirring. After30 min, a few more drops of bromine were added until the orange colourremained. Evaporation of the volatile materials gave a yellow foam.

Yield: quantitativeLC-MS (Method 2): Rt=3.82 min, m/z=508/510 [M+H]⁺

The following intermediates were prepared in a similar manner:

LC-MS Mass Precursor Rt (min) [M + H]⁺ Intermediate StructureIntermediate Yield (%) Method 1 17

8 94 3.78 474/476 18

9 100 3.72 550/552 [M + CH₃CN]⁺ 19

10 100 3.77 454/456 20

14 100 3.70 476/478 21

15 100 4.31 609/611

Intermediate 22

Intermediate 13 (32 mg, 0.029 mmol) was dissolved in chloroform (2 ml)and bromine (4 drops) was added. The solution was stirred at RT for 1 hafter which the volatiles were evaporated. The product was obtained as acream foam.

Yield: quantitativeLC-MS (Method 2): Rt=3.35 min, m/z=1182 [M]⁺

The following intermediates were prepared in a similar manner:

Pre- cur- sor LC- in- MS ter- Rt Mass me- Yield (min) Ion Structurediate (%) Method 2 23

 4 100 4.60 1338 [M + H]⁺ 24

 6  64 3.38 1128 [M + H]⁺ 25

 7 100 3.45 1142 [M]⁺ 26

11 100 3.36 1165 [M]⁺ 27

12 100 3.40 1168 [M]⁺ 28

 2 100 3.36 1228 [M]⁺

Intermediate 29

Intermediate 1 (1.00 g, 2.331 mmol) was dissolved in anhydrous DMF (25ml) and the solution was cooled to −10° C. under argon. Sodium hydride(60% dispersion in mineral oil) (93 mg, 2.331 mmol) was added and thereaction mixture was stirred until effervescence had ceased.N-Boc-3-bromopropylamine (610 mg, 2.563 mmol) was added and stirring wascontinued at 0° C. for a further 2.5 h, after which time sat. aqueousammonium chloride (60 ml) and EtOAc (60 ml) were added. The organiclayer was separated and the aqueous solution was further extracted withEtOAc (60 ml). The organic extracts were combined and washed with water(50 ml) and sat. brine (30 ml), dried (Na₂SO₄) and evaporated. Theresidue was purified on an Isolute™ Si II cartridge eluting with 0-30%EtOAc in pentane to give the product as a pale yellow oil.

Yield: 783 mg (57%)

LC-MS (Method 2): Rt=4.31 min, m/z=585 [M−H]⁻

Intermediate 30

Intermediate 29 (776 mg, 1.32 mmol) was dissolved in DCK/1 (20 ml) andN-bromosuccinimide (236 mg, 1.32 mmol) was added. The solution wasstirred at RT for 1.5 h and then the mixture was diluted with DCM (80ml), washed with sat. aqueous NaHCO₃ (50 ml), water (50 ml) and brine(30 ml), and dried (Na₂SO₄). Evaporation gave a pale yellow gum.

Yield: quantitativeLC-MS (Method 2): Rt=4.36 min, m/z=565/567 [M-Boc+2 H]⁺

Intermediate 31

Intermediate 30 (731 mg, 1.099 mmol) was dissolved in acetonitrile (20ml) and sodium hydrogen carbonate (277 mg, 3.297 mmol) and 2M ethylaminein THF (0.8 ml, 1.65 mmol) were added. The mixture was heated at 80° C.for 3.5 h, allowed to cool, filtered and evaporated. The residue waspartitioned between DCM (70 ml) and water (50 ml). The organic layer wasseparated and evaporated, and the crude product was purified on anIsolute™ Si II cartridge (10 g) eluting with 40-80% EtOAc in pentane togive the product as a cream foam.

Yield: 297 mg (46%)

LC-MS (Method 2): Rt=3.71 min, m/z=582 [M−H]⁻

Intermediate 32

Intermediate 31 (292 mg, 0.501 mmol) was dissolved in 20% TFA in DCM (20ml). After 2 h the volatiles were evaporated and the residue wasdissolved in MeOH and loaded onto an Isolute™ SCX-2 cartridge (5 g)which had been pretreated with MeOH. After flushing with MeOH, theproduct was eluted with 2M ammonia in MeOH. Evaporation of the UV activefractions gave the pure product as a pale yellow gum.

Yield: 221 mg (91%)

LC-MS (Method 2): Rt=2.33 min, m/z=484 [M+H]⁺

Intermediate 33

Intermediate 32 (214 mg, 0.443 mmol) was dissolved in DCM (10 ml) and1,1′-thiocarbonyidipyridone (51 mg, 0.222 mmol) was added. The solutionwas allowed to stand at RT for 48 h and then treated with a resin boundamine for 15 min. After filtering, the solvent was evaporated and thecrude product was purified on an Isolute™ Si II cartridge (5 g) elutingwith 0-5% MeOH in EtOAc. The fractions that contained product werecombined and evaporated, the residue dissolved in MeOH and passedthrough an Isolute™ SCX-2 cartridge (5 g), flushing further withmethanol. Evaporation gave a white foam.

Yield: 160 mg (36%)

LC-MS (Method 2): Rt=3.91 min, m/z=1009 [M+H]⁺

Intermediate 34

Intermediate 34 was prepared from Intermediate 1 and tert-butylbromoacetate using a similar procedure to that used in the synthesis ofIntermediate 29.

Yield: (80%)

LC-MS (Method 2): Rt=4.31 min, m/z=488 [M+H-tBu]⁺

Intermediate 35

Intermediate 35 was prepared from Intermediate 34 using a similar methodto that used in the preparation of Intermediate 30.

Yield: (41%)

LC-MS (Method 2): Rt=4.41 min, m/z=566/568 [M+H-tBu]⁻

Intermediate 36

A solution of Intermediate 35 (467 mg, 0.751 mmol) in acetonitrile (15ml) was treated with N,N-bis(3-aminopropyl)methylamine (54 mg, 0.375mmol) and sodium hydrogen carbonate (252 mg, 3.00 mmol). The reactionwas heated at 80° C. for 3.5 h. After allowing the mixture to cool, itwas filtered and the filtrate evaporated. Chromatography using anIsolute™ Si II cartridge (10 g), and eluting with 1-20% MeOH in EtOAc,gave the pure product as a white solid.

Yield: 182 mg (43%)

LC-MS (Method 2): Rt=3.22 min, m/z=1136 [M+H]⁺

Intermediate 37

Intermediate 36 (177 mg, 0.156 mmol) was dissolved in a mixture of DCM(30 ml) and iodomethane (8 ml). After standing at RT for 3 days thevolatiles were evaporated and the residue was taken up into acetonitrile(13 ml). lodomethane (4 ml) and sodium hydrogen carbonate (39 mg, 0.468mmol) were added and the mixture was heated at 80° C. under reflux.After 17 h the volatiles were evaporated and the residue was partitionedbetween DCM (50 ml) and water (50 ml). The organic phase was separatedand dried (Na₂SO₄). Evaporation gave a beige foam.

Yield: 181 mg (91%)

LC-MS (Method 2): Rt=3.20 min, m/z=1150 [M] ⁺

Intermediate 38

Intermediate 38 was prepared from Intermediate 16 and 0.5 equivalents of(2-aminoethyl){2-[(2-aminoethyl)tert-butoxycarbonylamino]ethyl}carbamicacid tert-butyl ester by a similar method to that used in the synthesisof intermediate 36.

Yield: (61%)

LC-MS (Method 2): Rt=3.58 min, m/z=1009 [M+H]⁺

Intermediate 39

The tert-butyloxycarbonyl protecting groups in Intermediate 39 wereremoved using a procedure analoaous to that described for thedeprotection of Intermediate 31.

Yield: (89%)

LC-MS (Method 2): Rt=2.17 min, m/z=909 [M+H]⁺

Intermediate 40

A solution of Intermediate 39 (219 mg, 0.241 mmol) and1,1′-thiocarbonyldipyridone (28 mg, 0.121 mmol) in DCM (10 ml) wasallowed to stand at RT for 24 h. A further portion of1,1′-thiocarbonyldipyridone (20 mg, 0.086 mmol) was added and, after 4h, the reaction mixture was treated with an amine resin. The mixture wasstirred for 15 min, filtered and loaded into an Isolute™ SCX-2 cartridge(10 g) which had been conditioned with MeOH. The cartridge was flushedwith MeOH and the eluent was evaporated. The white solid waschromatoaraphed on an Isolute™ Si II cartridge (10 g) eluting with 0-10%MeOH in EtOAc. Evaporation gave a white solid.

Yield: 150 mg (66%)

LC-MS (Method 2): Rt=3.00 min, m/z=951 [M+H]⁺

Intermediate 41

A solution of Example 23 (130 mg, 0.117 mmol) in triethylamine (0.5 ml)and DMF (0.5 ml) was degassed before (trimethylsilyl)acetylene (34 μl,0.235 mmol), copper (I) iodide (1.7 mg, 3 mol %) andbis(triphenylphosphine)palladium (II) chloride (8.4 ma, 5 mol %) wereadded then stirred and heated at 115° C. under argon for 2 h. The cooledmixture was poured into dilute sulphuric acid (25 ml) and extracted withEtOAc (2×25 ml). These extracts were washed with brine (10 ml) beforethe organic phase was isolated, dried (MgSO₄), filtered and concentratedin vacuo. Purification was achieved using an Isolute™ Si II cartridgeeluting with a 0-10% MeOH in DCM gradient. The product was isolated as acream solid.

Yield: 48 mg (39%)

LC-MS (Method 1): Rt3.36 min, m/z1050 [M+H]⁺

Intermediate 42

A solution of Intermediate 16 (200 mg, 0.394 mmol) and(3-aminobutyl)methylcarbamic acid tert-butyl ester (370 mg, 1.83 mmol)in acetonitrile (10 ml) was heated at 40° C. for 2 h. The solvent wasevaporated and the residue was dissolved in MeOH and loaded onto anIsolute™ SCX-2 cartridge (5 g) which had been conditioned with MeOH. Theproduct was flushed off with MeOH.

Yield: 225 mg (98%)

LC-MS (Method 2): Rt=3.63 min, m/z=484 [M+H-Boc]⁺

Intermediate 43

Intermediate 42 was deprotected in a similar manner to Intermediate 31.

Yield: (94%)

LC-MS (Method 2): Rt=2.11 min, m/z=484 [M+H]⁺

Intermediate 44

Intermediate 3 (219 mg, 0.379 mmol) was dissolved in acetonitrile (13ml) and a 2M solution of methylamine in THF (2 ml) was added. Thesolution was heated at 50° C. for 3 h and then the solution was allowedto stand at RT for 3 days. The volatiles were evaporated and the residuewas partitioned between DCM (100 ml) and water (80 ml). The organiclayer was separated and dried (Na₂SO₄). Evaporation gave a white foam.

Yield: 170 mg (85%)

LC-MS (Method 2): Rt=2.67 min, m/z=529 [M+H]⁺

Intermediate 45

A solution of Intermediate 44 (165 mg, 0.313 mmol) and phenoxypropylbromide (67 mg, 0.313 mmol) in acetonitrile (10 ml) was treated withsodium hydrogen carbonate (53 mg, 0.626 mmol) and the reaction mixturewas heated at 80° C. for 4 days. The solution was decanted onto anIsolute™ SCX-2 cartridge (5 g) which had been conditioned with MeOH. Thecartridge was flushed with MeOH and then the product was eluted with 2MNH₃ in MeOH. Evaporation gave a colourless gum.

Yield: 105 mg (51%)

LC-MS (Method 1): Rt=2.98 min, m/z=663 [M+H]⁺

Intermediate 46

A solution of Intermediate 45 (100 mg, 0.151 mmol) in chloroform (6 ml)was treated with bromine (10 μl). After 1 h a further portion of bromine(20 μl) was added. The volatiles were evaporated to give thedi-brominated product.

Yield: quantitativeLC-MS (Method 2): Rt=3.12 min, m/z=821 [M+H]⁺

Intermediate 47

A mixture of (4-fluoro-3-trifluoromethylphenyl)urea (WO2008/003412) (2.5g, 11.26 mmol), 4-cyanobenzaldehyde (1.77 g, 13.51 mmol), methylacetoacetate (1.57 g, 13.51 mmol) and polyphosphoric acid (6.5 g) in THF(45 ml) was heated at reflux under argon for 20h. The bulk of thesolvent was evaporated under reduced pressure and the residuepartitioned between water and ethyl acetate. The organic phase waswashed with water, aqueous potassium carbonate, water, and brine, dried(Na₂SO₄) and evaporated. The crude material was purified using aCombiFlash® companion (330 g cartridge) to yield the desired product asa pale yellow solid.

Yield: 3.03 g, 62%

LC-MS (Method 5): Rt=3.64 min, m/z=434 [M+H]⁺

Intermediate 48

A solution of bromine (0.23 ml. 4.5 mmol) in chloroform (2 ml) was addeddropwise to a solution of Intermediate 47 (1.86 g, 4.29 mmol) inchloroform (35 ml) at RT with stirring. After 1.5 h, the volatiles wereremoved under reduced pressure and the residue triturated with diethylether to give the product as a white solid.

Yield: 1.91 g, 87%

LC-MS (Method 5): Rt=3.76 min, m/z=512/514 [M+H]⁺

Intermediate 49

Intermediate 49 was prepared from (3-fluoro-5-trifluoromethylphenyl)urea(WO2008/003412), methyl acetoacetate and 4-cyanobenzaldehyde using amethod similar to that used for Intermediate 47.

Yield: 1.50 g, 51%

LC-MS (Method 5): Rt=3.68 min, m/z=434 [M+H]⁺

Intermediate 50

Intermediate 50 was prepared from intermediate 49 in an analogous manneras Intermediate 48.

Yield: 0.73 g, 77%

LC-MS (Method 5): Rt=3.78 min, m/z=512/514 [M+H]⁺

Intermediate 51

t-Butyl bromoacetate (1.42 g, 7.30 mmol) was added to a solution ofExample 3 (0.46 g, 0.487 mmol) in acetonitrile (15 ml) followed by DIPEA(0.94 g, 7.30 mmol) at RT. The reaction mixture was stirred at 50° C.for 17 h. The volatiles were removed under reduced pressure and theresidue triturated with diethyl ether (30 ml). The solid was collectedby filtration under suction and then partitioned between water and DCM.The organic layer was separated, dried (Na₂SO₄) and evaporated. Thecrude product was purified on an Isolute® SPE Si II cartridge (10 g)eluting with DCM, 10% MeOH in DCM and then 15% MeOH in DCM. Productcontaining fractions were combined and the solvent removed under reducedpressure to give the desired product as a cream coloured solid.

Yield: 0.35 g, 63%

LC-MS (Method 5): Rt=2.89 min, m/z=1058 [M]⁺

Example 1

To a solution of Intermediate 16 (200 mg, 0.394 mmol) in acetonitrile (5ml) were added a 2M solution of methylarnine in THF (197 μl, 0.394 mmol)and sodium hydrogen carbonate (165 mg, 1.97 mmol). The solution washeated at 80° C. for 16 h and than the mixture was filtered. The productwas purified by HPLC System 1 and the fractions containing pure materialwere combined and freeze dried. The product was obtained as a yellowsolid.

Yield: 62 mg (38%)

LCMS (Method 3): Rt=8.67 min, m/z=413 [M+H]⁺

1H NMR (400 MHz, DMSO-d6): δ=2.73 (s, 3H), 3.78 (d, 1H), 3.83 (d, 1H),5.44 (d, 1H), 7,67-7.82 (m, 5H), 7.85-7.90 (m, 2H), 7.91 (m, 1H), 8.19(d, 1H) ppm.

The following examples were prepared in a similar manner fromIntermediate 16 and an amine:

LC-MS (Method 1) Mass Example Structure Yield (%) Rt (min) [M + H]⁺ 2

50 9.11 427.04 3

 7 6.13 484.11 4

 5 8.03 399.00 5

27 6.39 526.09 6

62 10.40 455.04 7

37 6.43 507.01

Example 8

To a solution of hexamethylenediamine (0.197 mmol) in acetonitrile (5ml) were added Intermediate 16 (200 nig, 0.394 mmol) and sodium hydrogencarbonate (165 mg, 1.97 mmol) and the reaction mixture was heated to 80°C. for 16 h. The mixture was cooled to RT then the solution was filteredand the solvent evaporated. The crude product was purified by HPLCSystem 1. The product-containing fractions were combined and freezedried. The product was obtained as a white solid.

Yield: 50 mg (29%)

LCMS (Method 3): m/z=879 [M+H]⁺

1H NMR (400 MHz, DMSO-d8): δ=1.09 (br s, 4H); 1.29 (br s, 4H); 3.02-3.20(m, 4H); 3.79 (s, 4H); 5.44 (d, 2H); 7.68-7.92 (m, 16H); 8.19 (d, 2H)ppm.

In analogy to the procedure for Example 8, the following compounds wereprepared from the indicated intermediate and the appropriate diamine:

Pre- LC- Ex- cursor MS Mass am- Inter- Yield Rt [M + ple Structuremediate (%) (min) H]⁺  9

16  4 10.3  (Meth- od 3) 836.9 10

16 28 12.49 (Meth- od 3) 935.06 11

16 34 10.56 (Meth- od 30 983.02 12

16 29 11.09 (Meth- od 3) 966.96 13

16 15  7.86 (Meth- od 3) 879.99 14

16 28 10.26 (Meth- od 30 911.00 15

16 19  7.66 (Meth- od 3) 963.06 16

16 13  9.57 (Meth- od 3) 1012.95 17

16  6  8.02 (Meth- od 3) 908.04 18

16 55  8.08 (Meth- od 3) 894.48 19

17 61  7.56 (Meth- od 3) 840.48 20

18 54  7.31 (Meth- od 40 910.47 21

19 66  6.95 (Meth- od 4) 800.46 22

20 59  7.36 (Meth- od 3) 844.51 23

21 60  9.02 (Meth- od 3) 1110.38

Example 24

To a solution of Intermediate 28 in acetonitrile (5 ml) were addedNaHCO₃ (92 mg, 1.10 mmol) and 2M ethylamine in THF (220 pl, 0.44 mmol).The reaction mixture was heated at 80° C. for 3.5 h. A further amount of2M ethylamine in THF (220 μl, 0.44 mmol) was added and mixture washeated at 80° C. for 4 h. The mixture was filtered and the volatileswere evaporated. The crude product was purified using HPLC System 1 andthe fractions were combined and freeze-dried to give the product, whichwas purified further using HPLC System 1. The pure fractions werecombined and freeze-dried to give the product as a cream solid.

Yield: 16 mg (7%)

LC-MS (Method 3): Rt=8.69 min, m/z=1064.06 [M⁺]

The following examples were prepared in a similar manner:

Mass Precursor Yield LC-MS Rt [M + H]⁺ Structure Intermediate (%) (min)or [M]⁺ 25

24 49 2.70 (Method 2) 964 26

25  7 9.33 (Method 3) 978.22 27

26 42 8.92 (Method 4) 1001.50 28

27 37 8.99 (Method 4) 1004.52 29

22 25 9.05 (Method 4) 1018.53

Example 30

Intermediate 23 (195 mg, 0.146 mmol) was dissolved in acetonitrile (10ml) and NaHCO₃ (61 mg, 073 mmol) and 2M ethyiamine in THF (1.5 ml, 2.917mmol) were added. The reaction mixture was heated at 80° C. for 4 h andthen filtered and evaporated. The residue was dissolved in MeOH (12 ml)and a solution of K₂CO₃ (242 mg, 1.75 mmol) in water (5 ml) was added.The mixture was stirred at RT and after 30 min EtOAc (50 ml) and water(50 ml) were added. The organic solution was isolated, washed with brine(30 ml), dried (Na₂SO₄) and evaporated. The crude product was purifiedusing HPLC System 1 and freeze-dried to give the product as a pale creamsolid.

Yield: 49 mg (31%)

LC-MS (Method 3): Rt=8.96 min, m/z=1078.08 [M+H]⁺

Example 31

Tetra-n-butylammoniurn fluoride solution (1M in THF, 45 μl, 0.045 mmol)was added to a stirred solution of Intermediate 41 (45 mg, 0.043 mmol)in THF (2 ml) at RT. The solvent was removed in vacua after 1.5 h, water(25 ml) added and extracted with EtOAc (2×25 ml). These extracts werewashed with brine (10 ml) before the organic phase was isolated, dried(MgSO₄), filtered and concentrated in vacuo. Purification using anIsolute™ Si II cartridge, using a 0-10% MeOH in DCM gradient. gave acream solid. Further purification using HPLC System 2, followed byisolation using an SCX-2 cartridge washed through with MeOH beforeproduct was recovered with 2M ammonia in MeOH, afforded the titlecompound as an off-white solid.

Yield: 17 mg (43%)

LC-MS (Method 3): Rt 8.53 min, m/z 906.28 [M+H]⁺

Example 32

To a solution of Example 17 (50 mg, 0.055 mmol) in acetonitrile (2 ml)were added an excess of iodomethane (500 μL) and sodium hydrogencarbonate (14 mg, 0.16 mmol). The reaction mixture was stirred at RT for18 h then evaporated in vacuo. The crude product was purified by HPLCSystem 1. The product containing fractions were combined and freezedried.

Yield: 31 mg (54%)

LCMS (Method 3): m/z=922.08 [M]⁺

1H NMR (400 MHz, DMSO-d6): δ=171 (br m, 4H); 2.82 (s, 6H); 2.99-3.30 (m,8H); 3.81 (s, 4H); 5.40 (d, 2H); 7.61-7.90 (m, 16H); 8.21 (d, 2H) ppm.

The following examples were prepared using a similar procedure:

Yield LC-MS Rt Mass Structure Precursor (%) (min) [M]⁺ 33

Example 13 32 7.90 894.04 34

Example 19 57 7.44 (Method 3) 854.33 35

Example 21 45 7.26 (Method 3) 814.43 36

Example 22 69 7.30 (Method 3) 858.37 37

Example 23 40 8.94 (Method 3) 1124.18

Example 38

Intermediate 28 (347 mg, 0256 mmol) and N,N-dimethylethylenediamine (135mp, 1.536 mmol) were dissolved in acetonitrile (20 ml) and sodiumhydrogen carbonate (193 mg, 2.30 mmol) was added. The reaction mixturewas heated at 80° C. for 5 h after which time it was filtered andevaporated. The crude product was purified using HPLC System 1 and thepure fractions were combined and freeze-dried to give the bis-formatesalt as a pale cream solid.

Yield: 45 mg (14%)

LC-MS (Method 3): Rt=5.81 min, m/z=575.79 [M]²⁺/2

Example 39

Intermediate 33 (155 mg, 0.154 mmol) was dissolved in IMS (20 ml) andiodomethane (4 ml) was added. The solution was allowed to stand at RTfor 3 days. The volatiles were evaporated and the residue wasre-dissolved in 2M ammonia in EtOH (7 ml). The reaction was heated at50° C. for 48 h and concentrated, and the residue was purified usingHPLC System 1 and freeze-dried to give the product as a white solid.

Yield: 35 mg (23%)

LC-MS (Method 3): Rt=9.44 min, m/z=992.04 [M+H]⁺

Example 40

Example 40 was prepared from Intermediate 24 and(2-aminoethyl)trimethylammonium chloride hydrochloride by a similarmethod to that used in the synthesis of Example 38. The crude productwas purified using HPLC System 1 and freeze-dried to give a white solid.

Yield: (20%)

LC-MS (Method 3): Rt=6.17 min, m/z=539.86 [M]²⁺/2

Example 41

Intermediate 37 (175 mg, 0.137 mmol) was treated with a mixture of TPA(5 ml) and DCM (15 ml). The solution was allowed to stand at RT for 3 hand the volatiles were then evaporated. The residue was dissolved in asmall amount of DOM and diethyl ether was added. The cream solid thatprecipitated was filtered and dried.

Yield: 150 mg (95%)

LC-MS (Method 3): Rt=8.39 min, m/z=1038.09 [M]⁺

Example 42

Example 41 (130 mg, 0.133 mmol), N,N-dimethyiethylenediamine (30 mg,0.399 mmol) and IDIPEA (146 μl, 1.13 mmol) were dissolved in DMF (7 ml)and HATU (94 mg, 0.249 mmol) was added. The solution was allowed tostand at RT for 30 min and the DMF was evaporated. The residue wastreated with sat. aqueous sodium hydrogen carbonate (100 ml) andextracted with DCM (3×80 ml). Evaporation of the organic extracts gave apale yellow gum which was purified using HPLC System 1. The purefractions were freeze-dried to give the bis-formate salt as a whitesolid.

Yield: 96 mg (61%)

LC-MS (Method 3): Rt=5.99 min, m/z=589.75 [M]²⁺/2

Example 43

Example 43 was prepared from Intermediate 40 using a procedure similarto that used in the synthesis of Example 39. The product was purifiedusing HPLC System 1 and obtained as the formate salt.

Yield: (20%)

LC-MS (Method 3): Rt=8.03 min, m/z=934.47 [M+H]⁺

Example 44

Example 17 (6.28 g, 6.92 mmol) was dissolved in acetonitrile (100 ml)and a 30% solution of bromomethane in acetonitrile (60 ml) was added.The solution was heated at 80° C. in a sealed metal tube. After 24 h thesolvent was reduced to approximately half volume and then diluted withwater. The solution was freeze-dried to give a cream solid.

Yield: quantitativeLC-MS (Method 3): Rt=7.92 min, m/z=922.37 [M]⁺

1H NMR (400 MHz, DMSO-d6): δ=171 (br m, 4H); 2.82 (s, 6H); 2.99-3.30 (m,8H); 3.81 (s, 4H); 5.40 (d, 2H); 7.61-7.90 (m, 16H); 8.21 (d, 2H) ppm.

The following examples were prepared in a similar manner:

Yield LC-MS Rt Mass Structure Precursor (%) (min) [M]⁺ 45

Example 13 100 7.71 (Method 4) 894.19 46

Example 3 100 5.87 (Method 4) 498.28 47

Example 25  32 9.30 (Method 3) 978.42

Example 48

Example 17 (150 mg, 0.165 mmol) and 2-bromoethanol (420 mg, 1.65 mmol)were dissolved in acetonitrile (2 ml) and the solution was heated at 80°C. for 120 h. The volatiles were evaporated and the product was purifiedusing HPLC System 3. The pure fractions were combined and freeze-driedto give a cream solid.

Yield: 42 mg (25%)

LC-MS (Method 4): Rt=7.67 min, m/z=952.34 [M]⁺

The following examples were prepared using a similar procedure fromIntermediate 17 and an alkyl halide:

LC-MS Rt Mass Yield (min) [M + H]⁺ Structure (%) (Method 4) or [M]⁺ 49

13 8.52 966.18 50

20 7.62 965.33

Example 51

A solution of succinic acid (5.9 mg, 0.0499 mmol) in water (2 ml) wasadded to a tube containing silver (1) oxide (11.6 mg, 0.0499 mmol). Themixture was stirred in the dark for 17 h before a solution of Example 44(100 mg, 0.0997 mmol) in THF (2 ml) and acetonitrile (0.5 ml) was added.Stirring was continued for 3 days and then the mixture was filtered. Thefiltrate was evaporated and the residue was purified by HPLC System 3.The pure fractions were combined and freeze-dried to give a pale yellowsolid.

Yield: 29 mg (30%)

LC-MS (Method 4): Rt=7.70 min, m/z=922.33 [M]⁺1H NMR (400 MHz, MeOD): δ=1.84 (br m, 8H); 2.45 (4H, s); 2.92 (s, 12H);3.14 (m, 8H); 3.30 (m, 8H); 3.91 (m, partly exchanged with solvent);5.51 (s, 4H); 7.64-7.82 (m, 32H) ppm.

The following compounds were prepared in a similar manner:

LC- MS Mass Yield Rt [M + Structure (%) (min) H]⁺ 52

45 7.76 (Meth- od 4) 922.33 53

30 7.74 (Meth- od 4) 922.33 54

60 7.94 (Meth- od 3) 922.45 55

40 8.01 (Meth- od 3) 922.45 56

52 8.08 (Meth- od 3) 922.45

NMR Data: Example 52

1H NMR (400 MHz, MeOD): δ=1.84 (br m, 8H); 2.92 (s, 12H); 3.14 (m, 8H);3.30 (m, 8H); 3.91 (m, partly exchanged with solvent); 5.51 (s, 4H);6.60 (2H, s); 7.64-7.82(m, 32H) ppm.

Example 53

1H NMR (400 MHz, MeOD): δ=1.84 (br m, 8H); 2.92 (s, 12H); 3.14 (m, 8H);3.30 (m, 8H); 3.91 (m, partly exchanged with solvent); 5.51 (s, 4H);6.18 (2H, s); 7.64-7.82 (m, 32H) ppm.

Example 54

1H NMR (400 MHz, MeOD): δ=1.84 (br m, 8H); 2.92 (s, 12H); 3.14 (m, 8H);3.30 (m, 8H); 3.91 (m, partly exchanged with solvent); 4.22 (2H, s);5.51 (s, 4H); 7.64-7.82 (m, 32H) ppm.

Example 55

1H NMR (400 MHz, DMSO-d6): δ=1.71 (br m, 8H); 2.82 (s, 12H); 2.99-3.30(m, 16H); 3.81 (s, 8H); 5.40 (d, 4H); 7,34 (dd, 2H); 7.61-7.90 (m, 16H);8.12 (m, 4H); 8.81 (d, 2H) ppm.

Example 56

1H NMR (400 MHz, DMSO-d6): δ=1.71 (br m, 8H); 2.56 (s, 4H); 2.82 (s,12H); 2.99-3.30 (m, 16H); 3.81 (s, 8H); 5.40 (d, 4H); 7.61-7.90 (m,32H); 8.21 (d, 4H) ppm.

Example 57

Example 44 (100 mg, 0.0997 mmol) was dissolved in MeOH (50 ml) andloaded onto an Isolute™ SCX-2 cartridge which had been conditioned withMeOH. The cartridge was flushed with MeOH and then the product waseluted with 1.25M HCl in MeOH (60 ml). The solvent was evaporated andthe product was purified using HPLC System 3. The pure fractions werecombined and freeze-dried to give a white solid.

Yield: 35 mg (37%)

LC-MS (Method 4): Rt=7.72 min, m/z=922.22 [M]⁺

Example 58

Example 57 (956 mg, 0.998 mmol) was dissolved in acetonitrile (20 ml)and sodium tosylate (290 mg, 1.50 mmol) was added. The reaction mixturewas heated at 80° C. under argon for 17 h. After cooling, the solid wasfiltered off and the filtrate was evaporated. The product was purifiedon an Isolute™ Al-N cartridge (10 g) eluting with 0-6% MeOH in DOM, andobtained as a cream solid.

Yield: 5.03 (46%)

LC-MS ((Method 3): Rt=7.97 min, m/z=922.38 [M]⁺

1H NMR (400 MHz, DMSO-d6): δ=1.71 (br m, 4H); 2,24 (s, 3H); 2.82 (s,6H); 2.99-3.30 (m, 8H): 3.81 (s, 4H); 5.40 (d, 2H); 7.10 (d. 2H); 7.43(d, 2H); 7.61-7.90 (m, 16H); 8.21 (d, 2H) ppm. Example 59

Example 32 was passed through HPLC System 5. The pure fractions werecombined and freeze-dried to give an off-white solid.

LC-MS (Method 3): Rt=7.86 min, m/z=922.15 [M]⁺

1H NMR (400 MHz, DMSO-d6): δ=1.71 (br m, 4H); 2.82 (s, 6H); 2.99-3.30(m, 8H); 3.81 (s, 4H); 5.40 (d, 2H); 7.61-7.90 (m, 16H); 8.21 (d, 2H);8.27 (s, 1H) ppm. Example 60

Example 60 was prepared from Intermediate 43 using a method similar tothat used in the synthesis of Intermediate 45.

Yield: (36%)

LC-MS (Method 4): Rt=7.49 min, m/z=618.34 [M+H]⁺

Example 61

Example 61 prepared from Example 60 using a procedure similar to thatused in the synthesis of Example 44.

Yield: quantitative

LC-MS (Method 3): Rt=7.87 min, m/z=632.29 [M]⁺

Example 62

A solution of Intermediate 46 (124 mg, 0.151 mmol) in acetonitrile (6ml) was treated with a 2M solution of ethylamine in THF (755 μl, 1.51mmol). The solution was allowed to stand at RT for 17 h. Evaporation ofthe solvent gave a residue which was purified using HPLC System 2.

Yield: 24 mg (48%)

LC-MS (Method 4): Rt=8.74 min, m/z=738.35/740.30 [M+H]⁺

Example 63

Example 63 was prepared from Example 62 using a method analogous to thatused in the preparation of Example 44.

Yield: quantitativeLC-MS (Method 3): Rt=9.11 min, m/z=752.31/754.31 [M]⁺

Example 64

Example 64 was obtained during the synthesis of Example 60. Exchange ofthe counterion occurred during HPLC (System 2).

Yield: (9%)

LC-MS (Method 4): Rt=8.72 min, m/z=752.46 [M]⁺

Example 65

A solution of Example 44 (50 mg, 0.0499 mmol) in water (1 ml) and THF (1ml) was treated with silver (I) oxide (5.76 mg, 0.0248 mmol). After 18h, the mixture was filtered and the filtrate was treated with succinicacid (2.94 mg, 0.0248 mmol). After 1 h the mixture was purified usingHPLC System 4. The product was obtained a white solid.

Yield: 10 mg (20%)

LC-MS (Method 4): Rt=3.19 min, m/z=954.19 [M+H]⁺

Example 66

Example 18 (30 mg, 0.0335 mmol) and 2-bromoethanol (92 mg, 0.774 mmol)were dissolved in acetonitrile (5 ml) in the presence of sodiumcarbonate (62 mg, 0.774 mmol) and the reaction mixture was heated at 80°C. for 48 h under stirring. The suspension was filtered and concentratedand the residue purified using HPLC System 3. The fractions containingthe product were combined and freeze dried to afford a white fluffypowder.

Yield: 14 mg (39%)

LC-MS (Method 3): Rt=7.84 min, m/z=982.42 [M⁺]

Example 67

A solution of Intermediate 48 (0.45 g, 0.88 mmol) in anhydrous THF (10ml) was added dropwise to a solution ofN-(3-aminopropyl)-1,3-propanediamine (0.115 g, 0.88 mmol) andtriethylamine (0.356 g, 3.52 mmol) in anhydrous THF (5 ml) at roomtemperature with stirring. After 2 h, the volatiles were removed underreduced pressure and the residue partitioned between water and ethylacetate. The organic layer was separated, washed with water and brine,dried (Na₂SO₄) and evaporated.

The crude product was purified on an Isolute® SPE Si II cartridge (5 g)eluting with DCM, 10% MeOH in DCM and then 18% MeOH in DCM. Purefractions were combined and the solvent removed under reduced pressure.The residue was dissolved in 1:1 MeCN/H₂O (10 ml) and then freeze-driedto give the desired product as a white solid.

Yield: 0.122 g, 30%.

LC-MS (Method 6): Rt=8.09 min, m/z=930 [M+H]⁺

Example 68

Example 68 was prepared from Intermediate 50 using a similar procedureto that used in the synthesis of Example 67.

Yield: 0.151 g, 28%

LC-MS (Method 6): Rt=8.15 min, m/z=930 [M+H]⁺

Example 69

Example 69 was prepared from Intermediate 48 (1.50 g, 2.93 mmol) and3,3′-diamino-N-methyldipropylamine (0.319 g, 2.20 mmol), using a similarprocedure to that employed in the synthesis of Example 67.

Yield: 1.25 g, 90%

LC-MS (Method 6): Rt=8.15 min, m/z=944 [M+H]⁺

Example 70

TFA (5 ml) was added to a solution of intermediate 51 (0.34 g. 0.30mmol) in DCM (8 ml) at RT with stirring. After 20h the volatiles wereevaporated. The residue was partitioned between sat. aqueous sodiumhydrogen carbonate and ethyl acetate. The organic layer washed withbrine, dried (Na₂SO₄) and evaporated. The crude material was purified byHPLC (System 1) and after freeze-dry, the desired product was obtainedas a white solid.

Yield: 155 mg, 55%

LC-MS (Method 6): Rt=9.14 min, m/z=1002 [M+H]⁺

Example 71

A solution of 2-phenylethylamine (52 mg, 0.43 mmol) and triethylamine(87 mg, 0.86 mmol) in anhydrous THF (3 ml) was added to a solution ofIntermediate 48 (200 mg, 0.39 mmol) in anhydrous THF (5 ml) at roomtemperature with stirring. After 20 h, the volatiles were removed underreduced pressure and the residue partitioned between 1N aqueous HCl andethyl acetate. The organic layer was separated, washed with water,brine, dried (Na₂SO₄) and evaporated. The crude product was purified onan Isolute® SPE Si II cartridge (5 g) eluting with DCM, 20% EtOAc in DCMand 5% MeOH in DCM. Pure fractions were combined and the solvent removedunder reduced pressure to give the desired product as a white solid.

Yield: 197 mg, 97%.

LC-MS (Method 6): Rt=11.02 min, m/z=521 [M+H]⁺

Example 72

By analogy to the procedure of Example 8 of WO2007/129060, and usingIntermediate 20 therein as precursor, the above compound was prepared;yield 59%; LC-MS 7.36 (Method 3 in that WO publication); mass 844.51.

Example 73

By analogy to the procedure of Example 32 of WO2007/129060, and usingthe compound of Example 6 as precursor, the above compound was prepared;yield 69%; LC-MS 7.30 (Method 3 in that WO publication); mass 858.37.

Biological Assays

Compounds of the invention were tested for their HNE inhibitoryactivity.

Fluorescent Peptide Substrate

Assays were performed in 96-well plates at a total assay volume of 100μl. The final concentration of the enzyme (human leukocyte elastase,Sigma E8140) was 0.00036 units/well. A peptide substrate(MeO-Suc-Ala-Ala-Pro-ValAMC, Calbiochem #324745) was used, at the finalconcentration of 100 μM. The final concentration of DMSO was 1% in theassay buffer (0.05M Tris.HCl, pH 7.5, 0.1M NaCl; 0.1M CaCl2; 0,0005%brij-35).

The enzymatic reaction was started by adding the enzyme. The enzymaticreaction was performed at RT and after 30 mins stopped by adding 50 μlsoybean trypsin inhibitor (Sigma T-9003) at a final concentration of 50μg/well. Fluorescence was read on the FLEXstation (Molecular Devices)using 380 nm excitation and 460 nm emission filters. The potency of thecompounds was determined from a concentration series of 10concentrations in range from 1000 nM to 0.051 nM. The results are meansof two independent experiments, each performed in duplicate.

Using Fluorescently Labelled Elastin

Assays were performed in 96-well plate at a total assay volume of 100μl. The final concentration of the enzyme (human leukocyte elastase,Sigma E8140) was 0.002 units/well. Fluorescently labelled, solubilisedelastin from bovine neck ligament (Molecular Probes, E-12056) was usedat the final concentration of 15 μg/ml. The final concentration of DMSOwas 2.5% in the assay buffer (0.1M Tris-HCL, pH8.0, containing 0.2 mMsodium azide).

The enzymatic reaction was started by adding the enzyme. The enzymaticreaction was performed at RT and read after 120 minutes. Fluorescencewas read on the FLEXstation (Molecular Devices) using 485 nm excitationand 530 nm emission filters. The potency of the compounds was determinedfrom a concentration series of 10 concentrations in range from 25000 nMto 1 nM. The results are means of two independent experiments, eachperformed in duplicate.

All compounds of the Examples except Example 23 had IC50 values of lessthan 100 nm. Example 23 had an activity in the ranee 50-500nm.

HNE Induced Lung Haemorrhage in the Rat

Instillation of human neutrophil elastase (HNE) into rat lung causesacute lung damage. The extent of this injury can be assessed bymeasuring lung haemorrhage. Male Sprague Dawley rats (175-220g) wereobtained from Harlan UK Ltd., full barrier-bred and certified free fromspecified micro-organisms on receipt. Animals were weighed and randomlyassigned to treatment groups (7-12 animals per group).

The vehicle used was 1% DMSO/Saline. Inhibitors were dissolved in 1%DMSO before the addition of 0.9% saline.

Animals in each study used to determine the efficacy of the elastaseinhibitors delivered locally to the lung by a variety of routes. Ratswere anaesthetised with the inhaled anaesthetic isoflurane (4%) when thedose was given from 30 minutes to 6 h prior to human neutrophil elastase(NNE) administration or terminally anaesthetised withhypnorm:hypnovel:water (1.5:1:2 at 2.7 ml/kg) when the predose was givenat less than 30 minutes prior to HNE administration and dosed eitherintratracheally (i.t.) by transoral administration using a Penn Centuryrnicrosprayer or intranasally (i.n.) by dropping the fluid on to thenares. Animals either received vehicle or compound at a dose volume of0.5 ml/kg.

Animals that had been allowed to recover after dosing were terminallyanaesthetised with hypnorm:hypnovel:water (1.5:1:2 at 2.7 ml/kg). Oncesufficiently anaesthetised, HNE (600 units/ml) or sterile saline wasadministered by transoral tracheal instillation at a volume of 100 μlusing a Penn Century microsprayer. Animals were kept warm in atemperature controlled box and given top up doses of anaesthetic asrequired to ensure continuous anaesthesia until termination.

Animals were sacrificed (0.5 ml to 1 ml sodium pentobarbitone) one hourpost HNE challenge. The trachea was exposed and a small incision madebetween two tracheal rings allowing a cannula (10 gauge, O.D. 2-10 mm,Portex Ltd.) to be inserted approximately 2 cm into the trachea towardsthe lung. This was secured into place with a cotton ligature. The lungswere then lavaged (BAL) three times with fresh 4 ml aliquots ofheparinised (10 units/ml) phosphate buffered saline (PBS). The resultantBALF was kept on ice until it was centrifuged.

The BALF was centrifuged at 1000 r.p.m. for 10 minutes in a centrifugecooled to between 4 and 10° C. The supernatant was discarded and thecell pellet resuspended in 1 ml 0.1% CETAB/PBS to lyse the cells. Celllysates were frozen until spectrophotometric analysis for blood contentcould be made. Standards were prepared by making solutions of whole ratblood in 0.1% CETAB/PBS.

Once defrosted 100 μl of each lysed cell suspension was placed into aseparate well of a 96 well flat bottomed plate. All samples were testedin duplicate and 100 μl 0.1% CETAB/PBS was included on the plate as ablank. The OD of the contents of each well was measured at 415 nm usinga spectramax 250 (Molecular devices).

A standard curve was constructed by measuring the OD (at 415 nm) ofdifferent concentrations of blood in 0.1% CETAB/PBS (30, 10, 7, 3, 1,0.3, 0.1 μl/ml).

The amount of blood in each experimental sample was calculated bycomparison to the standard curve. Data were then analysed as below:

-   -   1) The mean OD for duplicates was calculated    -   2) The value for the blank was subtracted from the value for all        other samples    -   3) Data were assessed to evaluate the normality of distribution.

The compounds of Examples 17, 18, 26, 27, 30, 32, 40, 41, 42, 43, 49,59, 61 and 64 were tested in the above assay and were shown to beeffective in reducing the quantity of blood haemorrhaged relative tocontrol. For example, the compound of Example 32 showed a statisticallysignificant reduction in haemorrhage of 67% relative to control whenadministered at 30 mg/kg it., 1 hour prior to HINE.

1. A multimeric compound comprising two, three or four molecules,covalently linked through a linker framework, wherein the molecules haveformula (I):

wherein A is selected from the group consisting of aryl and heteroaryl;D is selected from the group consisting of oxygen and sulphur; R¹, R²,R³ and R⁵ are independently each selected from the group consisting ofhydrogen, halogen, nitro, cyano, C₁-C₆-alkyl, C₂-C₆-alkenyl,C₂-C₆-alkynyl, hydroxyl, C₁-C₆-alkoxy and C₂-C₆-alkenyloxy, whereinC₁-C₆-alkyl and C₁-C₆-alkoxy can be further substituted with one tothree identical or different radicals selected from the group consistingof halogen, hydroxy and C₁-C₄-alkoxy; R and R⁴ each independentlyrepresent a radical of formula⁻[X]_(m)-[Alk¹]_(p)-[Q]_(n)-[Alk²]_(q)-[X¹]_(k)-Z wherein k, m, n, p andq are independently selected from 0 and 1; Alk¹ and Alk² eachindependently represent an optionally substituted C₁-C₆ alkylene orC₂-C₆ alkenylene radical which may optionally contain an ether (—O—),thioether (—S—) or amino (—NR^(A)—) link wherein R^(A) is hydrogen orC₁-C₃ alkyl; Q represents (i) a divalent radical selected from the groupconsisting of —O—, —S—, —S(═O)—, —S(═O)₂—, —S⁺(R^(A))—,—N(R^(A))(R^(B))—, —C(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)NR^(A)—,—NR^(A)C(═O)—, —S(O₂)NR^(A)—, —NR^(A)S(O₂)—, —NR^(A)C(═O)NR^(B)—,—NR^(A)C(═NR^(A))NR^(B)—, —C(═NR^(D))NR^(E)—, and —NR^(E)C(═NR^(D))—,wherein R^(A), R^(B), R^(D) and R^(E) are independently, selected fromthe group consisting of hydrogen C₁-C₆ alkyl and C₃-C₆ cycloalkyl, orR^(A) and R^(B) or R^(D) and R^(E) taken together with the nitrogen towhich they are attached form a monocyclic heterocyclic ring of 5 to 7ring atoms which may contain a further heteroatom selected from N, O andS, or (ii) an optionally substituted divalent mono- or bicycliccarbocyclic or heterocyclic radical having 3-6 ring members; Xrepresents a divalent radical selected from the group consisting of—(C═O)—, —S(O₂)—, —C(═O)O—, —(C═O)NR^(A)— and —S(O₂)NR^(A)—, whereinR^(A) is selected from is the group consisting of hydrogen, C₁-C₆ alkyland C₃-C₆ cycloalkyl; X¹ is selected from the group consisting of —O—,—S—and —NH; and Z is hydrogen or an optionally substituted mono- orbicyclic carbocyclic or heterocyclic radical having 3-6 ring members,and pharmaceutically acceptable salts thereof.
 2. The multimericcompound as claimed in claim 1, wherein said two, three or fourmolecules are linked to the linker framework via their respectivenitrogen atoms shown in formula (I) as linked to R.
 3. The multimericcompound as claimed in claim 1, having the formula M-L-M¹ wherein L is adivalent linker radical and M and M¹ are each independently a radical offormula (IA) or (IB):


4. The multimeric compound as claimed in claim 3 wherein M and M¹ arethe same.
 5. The multimeric compound as claimed in claim 1, wherein thelinker framework or linker radical L is a divalent straight chain,saturated or unsaturated hydrocarbon radical having from 2 to 12 carbonatoms in the said chain, and wherein one or more carbons may be replacedby a divalent monocyclic or bicyclic carbocyclic or heterocyclic radicalhaving from 3 to 7 ring atoms in the or each ring, or by —O—, —S—,—S(═O)—, —S(═O)₂—, —C(═O)—, —N(R^(P))—, —N⁺(R^(P))(R^(Q))—, —C(═O)O—,—OC(═O)—, —C(═O)NR^(A)—, —NR^(A)C(═O)—, —S(O₂)NR^(A)—, —NR^(A)S(O₂)—,—NR^(A)C(═O)NR^(B)—, —NR^(A)C(═NR^(A))NR^(B)—, —C(═NR^(D))NR^(E)—, or—NR^(E)C(═NR^(D))—, wherein R^(A), R^(B), R^(D) and R^(E) areindependently hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl, and R^(P) andR^(Q) are independently hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl,HO—(C₁-C₆ alkyl)-, R^(A)R^(B)N—(C₁-C₆alkyl)-, or HOC(═O)—(C₁-C₆ alkyl)-,or R^(A)and R^(B), or R^(D) and R^(E), or R^(P) and R^(Q) taken togetherwith the nitrogens to which they are attached form a monocyclicheterocyclic ring of 5 to 7 ring atoms which may contain a furtherheteroatom selected from N, O and S; or wherein when one or more —(CH₂)—groups of the linker framework is or are replaced by a divalentmonocyclic or bicyclic carbocyclic or heterocyclic radical, the saidradical is selected from the following:


6. The compound as claimed in claim 5 wherein the linker framework L hasone of the following structures:


7. The compound as claimed in claim 1 having formula (X), (Y) or (Z):

wherein A⁻ is a pharmaceutically acceptable anion.
 8. A method oftreatment of a disease or condition in which HNE is implicated,comprising administering to a subject suffering such disease aneffective amount of a compound as claimed in claim 1,
 9. The method oftreatment according to claim 8, wherein the disease or condition isselected from the group consisting of chronic obstructive pulmonarydisease, chronic bronchitis, lung fibrosis, pneumonia, acute respiratorydistress syndrome, pulmonary emphysema, smoking-induced emphysema andcystic fibrosis.
 10. The method of treatment according to claim 8,wherein the disease or condition is selected from the group consistingof asthma, rhinitis, psoriasis, dermatitis (atopic and non-atopic).Crohn's disease, ulcerative colitis and irritable bowel disease.